CN117737016A - Aldolone reductase AKR1D1 mutant and encoding gene and application thereof - Google Patents
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Abstract
The invention relates to the technical field of biological enzyme engineering, in particular to an aldehyde ketone reductase AKR1D1 mutant, and a coding gene and application thereof. Compared with the wild aldehyde ketone reductase AKR1D1 with the amino acid sequence shown as SEQ ID NO. 2, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant provided by the invention is subjected to any one mutation of single mutation, two-two combined mutation, three combined mutation or four combined mutation at the 132 th position, 230 th position, 309 th position and 311 th position of the amino acid sequence shown as SEQ ID NO. 2. The aldehyde ketone reductase AKR1D1 and coenzyme regeneration system constructed by the invention can convert the substrate intermediate 1 into the key chiral intermediate 2 under the condition of room temperature, has mild reaction conditions, almost no byproducts, stable coenzyme circulation system and simple subsequent separation, and has wide industrialized application prospect.
Description
Technical Field
The invention relates to the technical field of biological enzyme engineering, in particular to an aldehyde ketone reductase AKR1D1 mutant, and a coding gene and application thereof.
Background
Obeticholic Acid (Obeticholic Acid), known as 3α,7α -dihydroxy-6α -ethyl-5β -cholanic Acid, belongs to a farnesol X receptor agonist, and indirectly inhibits the gene expression of cytochrome 7A1 (CYP 7 A1) by activating the farnesol X receptor, so that the synthesis of cholic Acid can be inhibited, and the method is used for treating primary biliary cirrhosis and nonalcoholic fatty liver disease.
Currently, obeticholic acid is prepared by chemical synthesis techniques, for example: patent document CN106749468A discloses a preparation method of obeticholic acid, which uses hyodeoxycholic acid HDCA as a starting material to sequentially perform esterification reaction, oxidation reaction, hydroxyl protection reaction, reaction with ethyl zinc bromide or diethyl zinc reagent, dehydration reaction, oxidation reaction, reduction reaction and reaction for removing hydroxyl and carboxyl protection to obtain obeticholic acid. The method not only can solve the problem of raw materials, but also can avoid severe reaction conditions such as strong alkalinity, high temperature and the like, and greatly improves the synthesis efficiency of the obeticholic acid, thereby providing a novel preparation method which has few byproducts, simple and convenient operation, mild reaction conditions and low cost and is suitable for mass production of the obeticholic acid.
Patent document CN108191939a discloses a method for preparing obeticholic acid intermediate and obeticholic acid, the synthetic route of the intermediate is:
the obeticholic acid intermediate has good stereoselectivity in the reaction process, greatly simplifies the synthesis difficulty of the obeticholic acid and reduces the synthesis cost of the obeticholic acid. The method has the advantages of safe raw materials, low cost and effective reduction of production cost.
Patent document CN110938106a discloses a method for preparing an obeticholic acid intermediate and obeticholic acid thereof, wherein the obeticholic acid intermediate is prepared by carbonyl reduction and hydroxyl protection by taking 3A-hydroxy-6-vinyl-7-oxo-cholanic acid as a raw material, and the obtained obeticholic acid intermediate contains two benzene ring structures, has strong ultraviolet absorption, is convenient for quality research by using an ultraviolet detector, ensures that the quality of the raw material in the last step is controllable, is in a solid state at room temperature, and can be refined by using conventional purification means such as recrystallization and the like; and then the reduction of palladium carbon is further used to obtain the obeticholic acid, and the reduction of the olefinic bond is finished firstly from the aspect of the reaction process, so that the follow-up reaction is convenient, the reaction yield is high, the conversion is complete, and the refining difficulty of the final product is reduced.
However, obeticholic acid or an obeticholic acid intermediate prepared by chemical synthesis technology has high environmental protection pressure, and particularly a 6-ethylene intermediate, as shown in formula I:
when obeticholic acid is obtained by reduction, selective reduction is required, and chiral reagents are expensive and heavy metals remain. According to the search, no case report exists for preparing the obeticholic acid or the obeticholic acid intermediate by an enzyme method at present.
Disclosure of Invention
In order to solve the defects of the prior art, the invention provides an aldehyde ketone reductase AKR1D1 mutant, and a coding gene and application thereof. The aldehyde ketone reductase AKR1D1 (AKR 1D 1) gene is an enzyme for reducing 4, 5-double bond of bile acid molecule A ring in organism, the position of the double bond is changed by protein directed evolution technology, the aldehyde ketone reductase AKR1D1 is changed into 6-position ethylene unsaturated double bond of reducible intermediate of formula I from reducing 4, 5-double bond, the changed aldehyde ketone reductase AKR1D1 mutant can obviously catalyze intermediate 1 to react to generate product 2, and the invention has good industrial application prospect. The specific reaction is as follows:
in order to solve the technical problems, the invention provides the following technical scheme:
the amino acid sequence of wild aldehyde ketone reductase AKR1D1 from human homosapiens is shown as SEQ ID NO. 2, and the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 1.
The gene sequence of the aldehyde ketone reductase AKR1D1 is synthesized by Changzhou basis biotechnology Co., ltd, the gene sequence is subjected to codon optimization aiming at the codon preference of escherichia coli, and NdeI and HindIII restriction enzyme sites are respectively added at two ends of a coding region. The target gene fragment is subjected to restriction enzyme NdeI and HindIII digestion, then is connected with a pET28a (+) vector (Novagen company) subjected to double digestion, is transformed and is screened, and positive plasmid AKR1D1-pET28a (+) obtained through screening is transferred into BL21 (DE 3) host bacteria, so that an in vitro heterologous expression system of AKR1D1 is constructed.
The construction of the aldehyde ketone reductase AKR1D1 mutant is obtained by a directional evolution technical means. The method comprises the following specific steps: the mutant is obtained by utilizing error-prone PCR, DNA rearrangement, semi-rational design, three-dimensional structure simulation and other directional proceeding technologies. More specifically, the method comprises the following steps: the invention predicts one or more possible active sites related to catalysis by utilizing the principle of energy minimization and molecular docking technology, then carries out site-directed mutagenesis on the active sites, and screens mutants with obviously improved activity.
Further, the sites of potential catalytic position change predicted by the molecular docking technology are Y132, W230, V309 and L311, site-directed mutagenesis is carried out on the four sites respectively, and High Pressure Liquid Chromatography (HPLC) is utilized for screening mutants. More specifically, (1) when tyrosine (Y) at position 132 is mutated to phenylalanine (F), the catalytic activity of the mutant is increased relative to the wild-type enzyme; (2) When tryptophan (W) at position 230 is mutated to alanine (a), mutant enzyme activity is increased relative to the wild-type enzyme; (3) When valine (V) at position 309 is mutated to leucine (L), mutant enzyme activity is increased relative to the wild-type enzyme; (4) When leucine (L) at position 311 is mutated to valine (V), the mutant enzyme activity is significantly improved. When the mutations at the 4 sites are combined in pairs or three or four, the catalytic activity of the mutant is greatly improved compared with that of a single mutant.
Therefore, the invention provides an aldehyde ketone reductase AKR1D1 mutant, which has an amino acid sequence which is subjected to any one mutation of single mutation, two-two combined mutation, three combined mutation or four combined mutation at the 132 th position, 230 th position, 309 th position and 311 th position of the amino acid sequence shown in SEQ ID NO. 2 compared with the wild aldehyde ketone reductase AKR1D1 with the amino acid sequence shown in SEQ ID NO. 2.
Further, the single mutation of the aldehyde ketone reductase AKR1D1 mutant is:
when the 132 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to phenylalanine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 4;
or when the 230 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tryptophan to alanine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 6;
or when the 309 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from valine to leucine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 8;
or when the 311 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from leucine to valine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 10.
Further, the three combined mutations of the aldehyde ketone reductase AKR1D1 mutant are:
when the 132 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to phenylalanine, the 309 th site is mutated from valine to leucine, the 311 th site is mutated from leucine to valine, and the amino acid sequence of the aldehyde ketoreductase AKR1D1 mutant is shown in SEQ ID NO. 12.
Further, the four combined mutations of the aldehyde ketone reductase AKR1D1 mutant are:
when the 132 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to phenylalanine, the 230 th site is mutated from tryptophan to alanine, the 309 th site is mutated from valine to leucine, the 311 th site is mutated from leucine to valine, and the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 14.
In addition, the invention also provides a coding gene of the aldehyde ketone reductase AKR1D1 mutant.
Specifically, the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 4 is shown as SEQ ID NO. 3;
or the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 6 is shown as SEQ ID NO. 5;
or the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 8 is shown as SEQ ID NO. 7;
or the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 10 is shown as SEQ ID NO. 9;
or the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 12 is shown as SEQ ID NO. 11;
or the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 14 is shown as SEQ ID NO. 13.
According to the prior public knowledge, any gene is connected into various expression vectors after being operated or transformed, is transformed into a proper host cell, and can over-express target protein through induction under proper conditions.
Furthermore, the invention also claims a vector containing the coding gene.
Specifically, the vector may be any of various expression vectors including, but not limited to, pET expression vector, pCW expression vector, pUC expression vector, or pPIC9k expression vector.
Furthermore, the invention also claims host cells containing the coding genes.
In particular, the host cell may be any suitable host cell including, but not limited to, E.coli, pichia pastoris, streptomyces, or Bacillus subtilis.
In addition, the invention also provides application of the aldehyde ketone reductase AKR1D1 mutant, coding genes, vectors and host cells in preparation of the obeticholic acid chiral intermediate-2, which comprises the following specific steps: the aldehyde ketone reductase AKR1D1 mutant, the coding gene, the vector and the host cell are used as biocatalysts to transform substrates (intermediates 1) to generate products (intermediates 2).
Further, the invention also provides a method for preparing the obeticholic acid chiral intermediate-2, which comprises the following steps of:
s1, configuring a reaction system, which comprises the following steps: 1-10 g/L of aldehyde ketone reductase AKR1D1 mutant, 50-200 mmol/L of sodium phosphate buffer solution with pH of 6.0-8.0, 0.1-0.5 g/L of coenzyme NADP+, 1-10 g/L of obeticholic acid chiral intermediate-1, 0.5-5 g/L of glucose, 1g/L of glucose dehydrogenase, and regulating the pH of a reaction system to 6.0-8.0; controlling the temperature of the reaction system to be 30 ℃ and stirring for reaction;
s2, performing HPLC detection after reacting for 24 hours to obtain the obeticholic acid chiral intermediate-2.
The enzyme for performing biocatalytic reaction in the method for preparing the obeticholic acid chiral intermediate-2 provided by the invention comprises pure enzyme, corresponding recombinant resting cells, crude enzyme liquid or crude enzyme powder and other existing forms.
In summary, compared with the prior art, the invention has the following beneficial effects:
the aldehyde ketone reductase AKR1D1 and coenzyme regeneration system constructed by the invention can convert the substrate intermediate 1 into the key chiral intermediate 2 under the condition of room temperature, has mild reaction conditions, almost no byproducts, stable coenzyme circulation system and simple subsequent separation, and has wide industrialized application prospect.
Detailed Description
The present invention will be described in further detail with reference to the following examples, which are not intended to limit the present invention, but are merely illustrative of the present invention. The experimental methods used in the following examples are not specifically described, but the experimental methods in which specific conditions are not specified in the examples are generally carried out under conventional conditions, and the materials, reagents, etc. used in the following examples are commercially available unless otherwise specified.
In the examples, the experimental procedures, which are not specified in particular conditions, are generally carried out according to conventional conditions, such as those described in the guidelines for molecular cloning experiments (J. Sambrook, D.W. Lassel, huang Peitang, wang Jiaxi, zhu Houchu, et cetera, third edition, beijing: science Press, 2002).
Example 1 construction of prokaryotic expression System
The AKR1D1 (aldehyde ketone reductase AKR1D 1) gene fragment was synthesized by Changzhou-ary biotechnology Co., ltd, and the gene sequence was codon optimized for the codon preference of E.coli and recombined onto the PUC57 vector. After double digestion with restriction enzymes NdeI and HindIII (available from New EnglandBiolabs, NEB) for 4h at 37℃1% agarose gel electrophoresis was separated and gel-cut recovered (gel recovery kit available from Tiangen Biochemical technologies (Beijing)). Followed by ligation overnight at 16℃with the expression vector pET28a (+) (Novagen) subjected to the same double cleavage under the action of T4 DNA ligase (available from Takara). DH5 alpha competent cells (purchased from Tiangen Biochemical technology (Beijing)) were transformed with the ligation solution, and colony PCR screening and sequencing verification were performed to obtain a positive recombinant plasmid AKR1d1-pET28a (+).
The positive recombinant plasmid AKR1D1-pET28a (+) is transformed into expression host bacterium BL21 (DE 3) (purchased from Tiangen Biochemical technology (Beijing) limited company) to obtain prokaryotic expression strain AKR1D1-pET28a (+)/BL 21 (DE 3) which is used as a primary strain for subsequent directed evolution and fermentation.
Example 2 shake flask fermentative preparation of enzymes
The expression strain AKR1D1-pET28a (+)/BL 21 (DE 3) constructed in example 1 was cultured overnight at 37℃with shaking in 5mL of LB liquid medium [10g/L tryptone (OXIO), 5g/L yeast powder (OXIO), 10g/L sodium chloride (Guog reagent) ] added with kanamycin sulfate at a final concentration of 30. Mu.g/mL, and then inoculated in 500mL of LB liquid medium containing kanamycin sulfate at a final concentration of 30. Mu.g/mL at 1% (V/V) at 200rpm, followed by shaking at 37 ℃. When the OD600 is between 0.8 and 1.0, the inducer IPTG (isopropyl-. Beta. -D-thiogalactoside, IPTG) is added at a final concentration of 0.1mmol/L and induced overnight at 25 ℃. The thalli are collected by centrifugation at the temperature of 4 ℃ and at the speed of 8000rpm, then are suspended in 50mM sodium phosphate buffer solution with pH of 7.0, are crushed by ultrasound (200W, 3s/5s,20 min), are centrifuged at the temperature of 4 ℃ and at the speed of 12000rpm for 20min, and the supernatant is taken for freeze drying, thus obtaining crude enzyme powder.
Example 3 construction and screening of mutants
1. Construction of the mutant:
site-directed mutagenesis was performed on four sites 132, 230, 309, 311 using the AKR1D1-pET28a (+) recombinant plasmid as a template (for specific mutagenesis operations, reference is made to Stratagene Corp.)Site-Directed Mutagenesis Kit description of operation). Wherein:
(1) 132 site mutation
Forward primer (SEQ ID NO: 15):
CAAACCGGGTGATGAGATCTTTCCACGTGATGAGAACGG,
reverse primer (SEQ ID NO: 16):
CCGTTCTCATCACGTGGAAAGATCTCATCACCCGGTTTG;
(2) 230 site mutation
Forward primer (SEQ ID NO: 17):
GTACTTCTCGTAACCCGATCGCGGTTAACGTCTCTTCTCCAC,
reverse primer (SEQ ID NO: 18):
GTGGAGAAGAGACGTTAACCGCGATCGGGTTACGAGAAGTAC;
(3) 309 site mutation
Forward primer (SEQ ID NO: 19):
TGAACAAGAACGTACGCTTCCTAGAACTGCTGATGTGGCGT,
reverse primer (SEQ ID NO: 20):
ACGCCACATCAGCAGTTCTAGGAAGCGTACGTTCTTGTTCA;
(4) 311 site mutation
Forward primer (SEQ ID NO: 21):
GAACGTACGCTTCGTAGAAGTGCTGATGTGGCGTGATCAT,
reverse primer (SEQ ID NO: 22):
ATGATCACGCCACATCAGCACTTCTACGAAGCGTACGTTC。
2. mutant culture:
after transforming BL21 (DE 3) host bacteria with the above-obtained plasmid, the plasmid was spread on LB solid medium containing 30. Mu.g/mL kanamycin, and cultured upside down at 37℃overnight, followed by picking up the monoclonal culture from the plate overnight. The overnight cultured bacterial liquid was transferred to an Erlenmeyer flask containing fresh LB medium, and after shaking culture at 37℃and 200rpm for 4 hours, IPTG was added to the resulting culture to give a final concentration of 0.1mmol/L for induction, and the resulting culture was incubated overnight at 25 ℃. The cells were collected by centrifugation at 8000rpm for 10min at 4℃and suspended in 50mmol/L sodium phosphate buffer pH7.0, followed by ultrasonication and screening.
3. Screening of mutants:
5g/L substrate concentration, 0.2g/LNADP+,10g/L glucose, 50mmol/L sodium phosphate buffer pH7.0, 1g/L glucose dehydrogenase (self-made), 10% of the prepared cell disruption solution were added, and the mixture was subjected to shaking reaction at 30℃and 220 rpm. Samples were taken at 2h and 24h, respectively, for HPLC detection and the unit enzyme activity for substrate intermediate 1 was calculated.
Clones with significantly improved substrate conversion rates at both 2h and 24h were subjected to extensive culture and then sequenced to verify mutation status. Sequencing results show that the mutant enzyme activity is significantly improved, and the mutation sites contained in the clone are as follows: tyrosine (Y) at position 132 is mutated to phenylalanine (F), tryptophan (W) at position 230 is mutated to alanine (a), valine (V) at position 309 is mutated to leucine (L), and leucine (L) at position 311 is mutated to valine (V).
And then carrying out three combined mutations and four combined mutations on the 4 sites, wherein the activity detection shows that the catalytic activity of the combined mutations of certain sites is obviously improved compared with that of single-point mutations, and the specific enzyme activity values are shown in the table 1 below.
Table 1 enzyme Activity of different combinations of mutations
1U relative to the amount of enzyme required to produce 1. Mu. Mol of product in 1 min.
Wherein:
when the 132 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to phenylalanine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 4, and correspondingly, the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 3.
When the 230 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tryptophan to alanine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 6, and correspondingly, the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 5.
When the 309 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from valine to leucine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 8, and correspondingly, the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 7.
When the 311 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from leucine to valine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 10, and correspondingly, the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 9.
When the 132 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to phenylalanine, the 309 th site is mutated from valine to leucine, the 311 th site is mutated from leucine to valine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 12, and correspondingly, the nucleotide sequence of the encoding gene is shown in SEQ ID NO. 11.
When the 132 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to phenylalanine, the 230 th site is mutated from tryptophan to alanine, the 309 th site is mutated from valine to leucine, the 311 th site is mutated from leucine to valine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown as SEQ ID NO. 14, and correspondingly, the nucleotide sequence of the encoding gene is shown as SEQ ID NO. 13.
The specific sequence is as follows:
(1) Wild-type aldehyde ketoreductase AKR1D1 encoding gene nucleotide sequence (SEQ ID NO: 1):
ATGGACCTGTCTGCAGCTAGCCACCGTATTCCACTGTCTGACGGTAACTCCATTCCGATCATCGGTCTGGGTACCTACTCTGAACCGAAATCCACTCCAAAGGGTGCTTGTGCTACCTCTGTTAAAGTTGCGATCGATACCGGTTATCGTCACATCGACGGTGCTTACATCTACCAGAACGAACACGAAGTTGGTGAAGCTATCCGTGAGAAGATCGCGGAAGGTAAAGTT CGTCGTGAAGACATCTTCTACTGCGGTAAACTGTGGGCTACCAACCACGTTCCGGAAATGGTTCGTCCAACTCTGGAACGTACCTTGCGTGTTCTGCAGCTGGACTACGTTGACCTGTACATCATCGAAGTTCCAATGGCGTTCAAACCGGGTGATGAGATCTATCCACGTGATGAGAACGGCAAATGGCTGTACCACAAATCCAACCTGTGCGCAACTTGGGAAGCGATGGAAGCGTGCAAAGACGCTGGTCTGGTTAAGTCTCTGGGTGTTTCCAACTTCAACCGTCGTCAGCTGGAACTGATCTTGAACAAACCGGGTCTGAAACACAAGCCGGTTTCTAACCAGGTTGAATGCCATCCGTACTTCACTCAGCCAAAGCTGCTGAAGTTCTGTCAGCAGCATGACATCGTTATCACCGCTTACAGTCCACTGGGTACTTCTCGTAACCCGATCTGGGTTAACGTCTCTTCTCCACCGCTGCTGAAAGATGCACTGCTGAACTCTCTGGGTAAACGTTACAACAAGACCGCTGCACAGATCGTTCTGCGTTTCAACATCCAGCGTGGTGTTGTTGTCATTCCGAAGTCTTTCAACCTTGAACGTATCAAAGAGAACTTCCAGATCTTCGACTTCTCTCTGACCGAAGAAGAGATGAAAGACATCGAAGCGCTGAACAAGAACGTACGCTTCGTAGAACTGCTGATGTGGCGTGATCATCCGGAATATCCGTTCCATGACGAATACTAA
(2) Wild-type aldehyde ketoreductase AKR1D1 amino acid sequence (SEQ ID NO: 2):
MDLSAASHRIPLSDGNSIPIIGLGTYSEPKSTPKGACATSVKVAIDTGYRHIDGAYIYQNEHEVGEAIREKIAEGKVRREDIFYCGKLWATNHVPEMVRPTLERTLRVLQLDYVDLYIIEVPMAFKPGDEIYPRDENGKWLYHKSNLCATWEAMEACKDAGLVKSLGVSNFNRRQLELILNKPGLKHKPVSNQVECHPYFTQPKLLKFCQQHDIVITAYSPLGTSRNPIWVNVSSPPLLKDALLNSLGKRYNKTAAQIVLRFNIQRGVVVIPKSFNLERIKENFQIFDFSLTEEEMKDIEALNKNVRFVELLMWRDHPEYPFHDEY
(3) Y132F mutant encoding gene nucleotide sequence (SEQ ID NO: 3):
ATGGACCTGTCTGCAGCTAGCCACCGTATTCCACTGTCTGACGGTAACTCCATTCCGATCATCGGTCTGGGTACCTACTCTGAACCGAAATCCACTCCAAAGGGTGCTTGTGCTACCTCTGTTAAAGTTGCGATCGATACCGGTTATCGTCACATCGACGGTGCTTACATCTACCAGAACGAACACGAAGTTGGTGAAGCTATCCGTGAGAAGATCGCGGAAGGTAAAGTTCGTCGTGAAGACATCTTCTACTGCGGTAAACTGTGGGCTACCAACCACGTTCCGGAAATGGTTCGTCCAACTCTGGAACGTACCTTGCGTGTTCTGCAGCTGGACTACGTTGACCTGTACATCATCGAAGTTCCAATGGCGTTCAAACCGGGTGATGAGATCTTTCCACGTGATGAGAACGGCAAATGGCTGTACCACAAATCCAACCTGTGCGCAACTTGGGAAGCGATGGAAGCGTGCAAAGACGCTGGTCTGGTTAAGTCTCTGGGTGTTTCCAACTTCAACCGTCGTCAGCTGGAACTGATCTTGAACAAACCGGGTCTGAAACACAAGCCGGTTTCTAACCAGGTTGAATGCCATCCGTACTTCACTCAGCCAAAGCTGCTGAAGTTCTGTCAGCAGCATGACATCGTTATCACCGCTTACAGTCCACTGGGTACTTCTCGTAACCCGATCTGGGTTAACGTCTCTTCTCCACCGCTGCTGAAAGATGCACTGCTGAACTCTCTGGGTAAACGTTACAACAAG ACCGCTGCACAGATCGTTCTGCGTTTCAACATCCAGCGTGGTGTTGTTGTCATTCCGAAGTCTTTCAACCTTGAACGTATCAAAGAGAACTTCCAGATCTTCGACTTCTCTCTGACCGAAGAAGAGATGAAAGACATCGAAGCGCTGAACAAGAACGTACGCTTCGTAGAACTGCTGATGTGGCGTGATCATCCGGAATATCCGTTCCATGACGAATACTAA
(4) Y132F mutant amino acid sequence (SEQ ID NO: 4):
MDLSAASHRIPLSDGNSIPIIGLGTYSEPKSTPKGACATSVKVAIDTGYRHIDGAYIYQNEHEVGEAIREKIAEGKVRREDIFYCGKLWATNHVPEMVRPTLERTLRVLQLDYVDLYIIEVPMAFKPGDEIFPRDENGKWLYHKSNLCATWEAMEACKDAGLVKSLGVSNFNRRQLELILNKPGLKHKPVSNQVECHPYFTQPKLLKFCQQHDIVITAYSPLGTSRNPIWVNVSSPPLLKDALLNSLGKRYNKTAAQIVLRFNIQRGVVVIPKSFNLERIKENFQIFDFSLTEEEMKDIEALNKNVRFVELLMWRDHPEYPFHDEY
(5) The W230A mutant encoding gene nucleotide sequence (SEQ ID NO: 5):
ATGGACCTGTCTGCAGCTAGCCACCGTATTCCACTGTCTGACGGTAACTCCATTCCGATCATCGGTCTGGGTACCTACTCTGAACCGAAATCCACTCCAAAGGGTGCTTGTGCTACCTCTGTTAAAGTTGCGATCGATACCGGTTATCGTCACATCGACGGTGCTTACATCTACCAGAACGAACACGAAGTTGGTGAAGCTATCCGTGAGAAGATCGCGGAAGGTAAAGTTCGTCGTGAAGACATCTTCTACTGCGGTAAACTGTGGGCTACCAACCACGTTCCGGAAATGGTTCGTCCAACTCTGGAACGTACCTTGCGTGTTCTGCAGCTGGACTACGTTGACCTGTACATCATCGAAGTTCCAATGGCGTTCAAACCGGGTGATGAGATCTATCCACGTGATGAGAACGGCAAATGGCTGTACCACAAATCCAACCTGTGCGCAACTTGGGAAGCGATGGAAGCGTGCAAAGACGCTGGTCTGGTTAAGTCTCTGGGTGTTTCCAACTTCAACCGTCGTCAGCTGGAACTGATCTTGAACAAACCGGGTCTGAAACACAAGCCGGTTTCTAACCAGGTTGAATGCCATCCGTACTTCACTCAGCCAAAGCTGCTGAAGTTCTGTCAGCAGCATGACATCGTTATCACCGCTTACAGTCCACTGGGTACTTCTCGTAACCCGATCGCGGTTAACGTCTCTTCTCCACCGCTGCTGAAAGATGCACTGCTGAACTCTCTGGGTAAACGTTACAACAAGACCGCTGCACAGATCGTTCTGCGTTTCAACATCCAGCGTGGTGTTGTTGTCATTCCGAAGTCTTTCAACCTTGAACGTATCAAAGAGAACTTCCAGATCTTCGACTTCTCTCTGACCGAAGAAGAGATGAAAGACATCGAAGCGCTGAACAAGAACGTACGCTTCGTAGAACTGCTGATGTGGCGTGATCATCCGGAATATCCGTTCCATGACGAATACTAA
(6) W230A mutant amino acid sequence (SEQ ID NO: 6):
MDLSAASHRIPLSDGNSIPIIGLGTYSEPKSTPKGACATSVKVAIDTGYRHIDGAYIYQNEHEVGEAIREKIAEGKVRREDIFYCGKLWATNHVPEMVRPTLERTLRVLQLDYVDLYIIEVPMAFKPGDEIYPRDENGKWLYHKSNLCATWEAMEACKDAGLVKSLGVSNFNRRQLELILNKPGLKHKPVSNQVECHPYFTQPKLLKFCQQHDIVITAYSPLGTSRNPIAVNVSSPPLLKDA LLNSLGKRYNKTAAQIVLRFNIQRGVVVIPKSFNLERIKENFQIFDFSLTEEEMKDIEALNKNVRFVELLMWRDHPEYPFHDEY
(7) V309L mutant encoding gene nucleotide sequence (SEQ ID NO: 7):
ATGGACCTGTCTGCAGCTAGCCACCGTATTCCACTGTCTGACGGTAACTCCATTCCGATCATCGGTCTGGGTACCTACTCTGAACCGAAATCCACTCCAAAGGGTGCTTGTGCTACCTCTGTTAAAGTTGCGATCGATACCGGTTATCGTCACATCGACGGTGCTTACATCTACCAGAACGAACACGAAGTTGGTGAAGCTATCCGTGAGAAGATCGCGGAAGGTAAAGTTCGTCGTGAAGACATCTTCTACTGCGGTAAACTGTGGGCTACCAACCACGTTCCGGAAATGGTTCGTCCAACTCTGGAACGTACCTTGCGTGTTCTGCAGCTGGACTACGTTGACCTGTACATCATCGAAGTTCCAATGGCGTTCAAACCGGGTGATGAGATCTATCCACGTGATGAGAACGGCAAATGGCTGTACCACAAATCCAACCTGTGCGCAACTTGGGAAGCGATGGAAGCGTGCAAAGACGCTGGTCTGGTTAAGTCTCTGGGTGTTTCCAACTTCAACCGTCGTCAGCTGGAACTGATCTTGAACAAACCGGGTCTGAAACACAAGCCGGTTTCTAACCAGGTTGAATGCCATCCGTACTTCACTCAGCCAAAGCTGCTGAAGTTCTGTCAGCAGCATGACATCGTTATCACCGCTTACAGTCCACTGGGTACTTCTCGTAACCCGATCTGGGTTAACGTCTCTTCTCCACCGCTGCTGAAAGATGCACTGCTGAACTCTCTGGGTAAACGTTACAACAAGACCGCTGCACAGATCGTTCTGCGTTTCAACATCCAGCGTGGTGTTGTTGTCATTCCGAAGTCTTTCAACCTTGAACGTATCAAAGAGAACTTCCAGATCTTCGACTTCTCTCTGACCGAAGAAGAGATGAAAGACATCGAAGCGCTGAACAAGAACGTACGCTTCCTAGAACTGCTGATGTGGCGTGATCATCCGGAATATCCGTTCCATGACGAATACTAA
(8) V309L mutant amino acid sequence (SEQ ID NO: 8):
MDLSAASHRIPLSDGNSIPIIGLGTYSEPKSTPKGACATSVKVAIDTGYRHIDGAYIYQNEHEVGEAIREKIAEGKVRREDIFYCGKLWATNHVPEMVRPTLERTLRVLQLDYVDLYIIEVPMAFKPGDEIYPRDENGKWLYHKSNLCATWEAMEACKDAGLVKSLGVSNFNRRQLELILNKPGLKHKPVSNQVECHPYFTQPKLLKFCQQHDIVITAYSPLGTSRNPIWVNVSSPPLLKDALLNSLGKRYNKTAAQIVLRFNIQRGVVVIPKSFNLERIKENFQIFDFSLTEEEMKDIEALNKNVRFLELLMWRDHPEYPFHDEY
(9) L311V mutant encoding gene nucleotide sequence (SEQ ID NO: 9):
ATGGACCTGTCTGCAGCTAGCCACCGTATTCCACTGTCTGACGGTAACTCCATTCCGATCATCGGTCTGGGTACCTACTCTGAACCGAAATCCACTCCAAAGGGTGCTTGTGCTACCTCTGTTAAAGTTGCGATCGATACCGGTTATCGTCACATCGACGGTGCTTACATCTACCAGAACGAACACGAAGTTGGTGAAGCTATCCGTGAGAAGATCGCGGAAGGTAAAGTTCGTCGTGAAGACATCTTCTACTGCGGTAAACTGTGGGCTACCAACCACGTTCCGGAAATGGTTCGTCCAACTCTGGAACGTACCTTGCGTGTTCTGCAGCTGGACTACGTTGACCTG TACATCATCGAAGTTCCAATGGCGTTCAAACCGGGTGATGAGATCTATCCACGTGATGAGAACGGCAAATGGCTGTACCACAAATCCAACCTGTGCGCAACTTGGGAAGCGATGGAAGCGTGCAAAGACGCTGGTCTGGTTAAGTCTCTGGGTGTTTCCAACTTCAACCGTCGTCAGCTGGAACTGATCTTGAACAAACCGGGTCTGAAACACAAGCCGGTTTCTAACCAGGTTGAATGCCATCCGTACTTCACTCAGCCAAAGCTGCTGAAGTTCTGTCAGCAGCATGACATCGTTATCACCGCTTACAGTCCACTGGGTACTTCTCGTAACCCGATCTGGGTTAACGTCTCTTCTCCACCGCTGCTGAAAGATGCACTGCTGAACTCTCTGGGTAAACGTTACAACAAGACCGCTGCACAGATCGTTCTGCGTTTCAACATCCAGCGTGGTGTTGTTGTCATTCCGAAGTCTTTCAACCTTGAACGTATCAAAGAGAACTTCCAGATCTTCGACTTCTCTCTGACCGAAGAAGAGATGAAAGACATCGAAGCGCTGAACAAGAACGTACGCTTCGTAGAAGTGCTGATGTGGCGTGATCATCCGGAATATCCGTTCCATGACGAATACTAA
(10) L311V mutant amino acid sequence (SEQ ID NO: 10):
MDLSAASHRIPLSDGNSIPIIGLGTYSEPKSTPKGACATSVKVAIDTGYRHIDGAYIYQNEHEVGEAIREKIAEGKVRREDIFYCGKLWATNHVPEMVRPTLERTLRVLQLDYVDLYIIEVPMAFKPGDEIYPRDENGKWLYHKSNLCATWEAMEACKDAGLVKSLGVSNFNRRQLELILNKPGLKHKPVSNQVECHPYFTQPKLLKFCQQHDIVITAYSPLGTSRNPIWVNVSSPPLLKDALLNSLGKRYNKTAAQIVLRFNIQRGVVVIPKSFNLERIKENFQIFDFSLTEEEMKDIEALNKNVRFVEVLMWRDHPEYPFHDEY
(11) Y132F/V309L/L311V mutant encoding gene nucleotide sequence (SEQ ID NO: 11):
ATGGACCTGTCTGCAGCTAGCCACCGTATTCCACTGTCTGACGGTAACTCCATTCCGATCATCGGTCTGGGTACCTACTCTGAACCGAAATCCACTCCAAAGGGTGCTTGTGCTACCTCTGTTAAAGTTGCGATCGATACCGGTTATCGTCACATCGACGGTGCTTACATCTACCAGAACGAACACGAAGTTGGTGAAGCTATCCGTGAGAAGATCGCGGAAGGTAAAGTTCGTCGTGAAGACATCTTCTACTGCGGTAAACTGTGGGCTACCAACCACGTTCCGGAAATGGTTCGTCCAACTCTGGAACGTACCTTGCGTGTTCTGCAGCTGGACTACGTTGACCTGTACATCATCGAAGTTCCAATGGCGTTCAAACCGGGTGATGAGATCTTTCCACGTGATGAGAACGGCAAATGGCTGTACCACAAATCCAACCTGTGCGCAACTTGGGAAGCGATGGAAGCGTGCAAAGACGCTGGTCTGGTTAAGTCTCTGGGTGTTTCCAACTTCAACCGTCGTCAGCTGGAACTGATCTTGAACAAACCGGGTCTGAAACACAAGCCGGTTTCTAACCAGGTTGAATGCCATCCGTACTTCACTCAGCCAAAGCTGCTGAAGTTCTGTCAGCAGCATGACATCGTTATCACCGCTTACAGTCCACTGGGTACTTCTCGTAACCCGATCTGGGTTAACGTCTCTTCTCCACCGCTGCTGAAAGATGCACTGCTGAACTCTCTGGGTAAACGTTACAACAAGACCGCTGCACAGATCGTTCTGCGTTTCAACATCCAGCGTGGTGTTGTTGTCATTCCGAAGTCTTTCAACCTTGAACGTATCAAAGAGAACTTCCAGATCTTCGACTTCTCTCTGACCG AAGAAGAGATGAAAGACATCGAAGCGCTGAACAAGAACGTACGCTTCCTAGAAGTGCTGATGTGGCGTGATCATCCGGAATATCCGTTCCATGACGAATACTAA
(12) Y132F/V309L/L311V mutant amino acid sequence (SEQ ID NO: 12):
MDLSAASHRIPLSDGNSIPIIGLGTYSEPKSTPKGACATSVKVAIDTGYRHIDGAYIYQNEHEVGEAIREKIAEGKVRREDIFYCGKLWATNHVPEMVRPTLERTLRVLQLDYVDLYIIEVPMAFKPGDEIFPRDENGKWLYHKSNLCATWEAMEACKDAGLVKSLGVSNFNRRQLELILNKPGLKHKPVSNQVECHPYFTQPKLLKFCQQHDIVITAYSPLGTSRNPIWVNVSSPPLLKDALLNSLGKRYNKTAAQIVLRFNIQRGVVVIPKSFNLERIKENFQIFDFSLTEEEMKDIEALNKNVRFLEVLMWRDHPEYPFHDEY
(13) Y132F/W230A/V309L/L311V mutant encoding gene nucleotide sequence (SEQ ID NO: 13):
ATGGACCTGTCTGCAGCTAGCCACCGTATTCCACTGTCTGACGGTAACTCCATTCCGATCATCGGTCTGGGTACCTACTCTGAACCGAAATCCACTCCAAAGGGTGCTTGTGCTACCTCTGTTAAAGTTGCGATCGATACCGGTTATCGTCACATCGACGGTGCTTACATCTACCAGAACGAACACGAAGTTGGTGAAGCTATCCGTGAGAAGATCGCGGAAGGTAAAGTTCGTCGTGAAGACATCTTCTACTGCGGTAAACTGTGGGCTACCAACCACGTTCCGGAAATGGTTCGTCCAACTCTGGAACGTACCTTGCGTGTTCTGCAGCTGGACTACGTTGACCTGTACATCATCGAAGTTCCAATGGCGTTCAAACCGGGTGATGAGATCTTTCCACGTGATGAGAACGGCAAATGGCTGTACCACAAATCCAACCTGTGCGCAACTTGGGAAGCGATGGAAGCGTGCAAAGACGCTGGTCTGGTTAAGTCTCTGGGTGTTTCCAACTTCAACCGTCGTCAGCTGGAACTGATCTTGAACAAACCGGGTCTGAAACACAAGCCGGTTTCTAACCAGGTTGAATGCCATCCGTACTTCACTCAGCCAAAGCTGCTGAAGTTCTGTCAGCAGCATGACATCGTTATCACCGCTTACAGTCCACTGGGTACTTCTCGTAACCCGATCGCGGTTAACGTCTCTTCTCCACCGCTGCTGAAAGATGCACTGCTGAACTCTCTGGGTAAACGTTACAACAAGACCGCTGCACAGATCGTTCTGCGTTTCAACATCCAGCGTGGTGTTGTTGTCATTCCGAAGTCTTTCAACCTTGAACGTATCAAAGAGAACTTCCAGATCTTCGACTTCTCTCTGACCGAAGAAGAGATGAAAGACATCGAAGCGCTGAACAAGAACGTACGCTTCCTAGAAGTGCTGATGTGGCGTGATCATCCGGAATATCCGTTCCATGACGAATACTAA
(14) Y132F/W230A/V309L/L311V mutant amino acid sequence (SEQ ID NO: 14):
MDLSAASHRIPLSDGNSIPIIGLGTYSEPKSTPKGACATSVKVAIDTGYRHIDGAYIYQNEHEVGEAIREKIAEGKVRREDIFYCGKLWATNHVPEMVRPTLERTLRVLQLDYVDLYIIEVPMAFKPGDEIFPRDENGKWLYHKSNLCATWEAMEACKDAGLVKSLGVSNFNRRQLELILNKPGLKHKPVSNQVECHPYFTQPKLLKFCQQHDIVITAYSPLGTSRNPIAVNVSSPPLLKDALLNSLGKRYNKTAAQIVLRFNIQRGVVVIPKSFNLERIKENFQIFDFSLTEEEMKDIEALNKNVRFLEVLMWRDHPEYPFHDEY
example 4 biocatalysis of mutants
0.1g of substrate (intermediate 1) was dissolved in 100mL of 50mmol/L sodium phosphate buffer solution with pH9.0, the pH was adjusted to 7.0 with liquid alkali, after the substrate was completely dissolved, 0.5g of glucose, 0.02g of NADP+, 1g of AKR1D1 mutant lyophilized powder (AKR 1D1 mutant lyophilized powder prepared in example 2) and 0.1g of glucose dehydrogenase lyophilized powder were added, the reaction solution was placed in a constant temperature water bath at 30℃and reacted with mechanical stirring, the pH of the system was controlled at 7.0 with 2mol/L sodium hydroxide solution, and after 24 hours of reaction, HPLC detection was performed, and the substrate conversion rate was as shown in Table 2.
TABLE 2 conversion of mutants
Amino acid sequence | Mutant | Conversion rate |
SEQ ID NO:2 | WildtypeAKR1D1 | <1% |
SEQ ID NO:4 | Y132F | 25.31% |
SEQ ID NO:6 | W230A | 15.12% |
SEQ ID NO:8 | V309L | 31.43% |
SEQ ID NO:10 | L311V | 28.45% |
SEQ ID NO:12 | Y132F/V309L/L311V | 52.31% |
SEQ ID NO:14 | Y132F/W230A/V309L/L311V | 72.89% |
Finally, it should be noted that the above description is only for illustrating the technical solution of the present invention, and not for limiting the scope of the present invention, and that the simple modification and equivalent substitution of the technical solution of the present invention can be made by those skilled in the art without departing from the spirit and scope of the technical solution of the present invention.
Claims (9)
1. The aldehyde ketone reductase AKR1D1 mutant is characterized in that compared with wild aldehyde ketone reductase AKR1D1 with an amino acid sequence shown as SEQ ID NO. 2, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is subjected to any one mutation of 132 rd, 230 th, 309 th and 311 th mutation, two-two combined mutation, three combined mutation or four combined mutation of the amino acid sequence shown as SEQ ID NO. 2.
2. The aldehyde ketone reductase AKR1D1 mutant according to claim 1, wherein the single mutation is:
when the 132 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to phenylalanine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 4;
or when the 230 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tryptophan to alanine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 6;
or when the 309 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from valine to leucine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 8;
or when the 311 th position of the amino acid sequence shown in SEQ ID NO. 2 is mutated from leucine to valine, the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 10.
3. The aldehyde ketone reductase AKR1D1 mutant according to claim 1, wherein the three combined mutations are:
when the 132 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to phenylalanine, the 309 th site is mutated from valine to leucine, the 311 th site is mutated from leucine to valine, and the amino acid sequence of the aldehyde ketoreductase AKR1D1 mutant is shown in SEQ ID NO. 12.
4. The aldehyde ketone reductase AKR1D1 mutant according to claim 1, wherein the four combined mutations are:
when the 132 th site of the amino acid sequence shown in SEQ ID NO. 2 is mutated from tyrosine to phenylalanine, the 230 th site is mutated from tryptophan to alanine, the 309 th site is mutated from valine to leucine, the 311 th site is mutated from leucine to valine, and the amino acid sequence of the aldehyde ketone reductase AKR1D1 mutant is shown in SEQ ID NO. 14.
5. The coding gene of the aldehyde ketone reductase AKR1D1 mutant as claimed in any one of claims 2 to 4, wherein the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown in SEQ ID NO. 4 is shown in SEQ ID NO. 3;
or the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 6 is shown as SEQ ID NO. 5;
or the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 8 is shown as SEQ ID NO. 7;
or the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 10 is shown as SEQ ID NO. 9;
or the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 12 is shown as SEQ ID NO. 11;
or the nucleotide sequence of the coding gene of the aldehyde ketone reductase AKR1D1 mutant with the amino acid sequence shown as SEQ ID NO. 14 is shown as SEQ ID NO. 13.
6. A vector containing the coding gene according to claim 5, wherein the vector is a pET expression vector, a pCW expression vector, a pUC expression vector or a pPIC9k expression vector.
7. A host cell comprising the coding gene of claim 5, wherein the host cell is E.coli, pichia pastoris, streptomyces or Bacillus subtilis.
8. Use of the aldehyde ketone reductase AKR1D1 mutant according to any one of claims 1 to 4, the coding gene according to claim 5, the vector according to claim 6, the host cell according to claim 7 for the preparation of obeticholic acid chiral intermediate-2.
9. A process for the preparation of obeticholic acid chiral intermediate-2 comprising the steps of:
s1, configuring a reaction system, which comprises the following steps: 1-10 g/L of the aldehyde ketone reductase AKR1D1 mutant as claimed in any one of claims 1-4, 50-200 mmol/L of sodium phosphate buffer solution with pH of 6.0-8.0, 0.1-0.5 g/L of coenzyme NADP+, 1-10 g/L of obeticholic acid chiral intermediate-1, 0.5-5 g/L of glucose, 1g/L of glucose dehydrogenase, and adjusting the pH of a reaction system to 6.0-8.0; controlling the temperature of the reaction system to be 30 ℃ and stirring for reaction;
s2, performing HPLC detection after reacting for 24 hours to obtain the obeticholic acid chiral intermediate-2.
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